DNA double-strand breaks (DSBs) are highly toxic to cells, and can be introduced by ionizing radiation (IR) or interstrand crosslinking agents, but also occur spontaneously during DNA replication. If mis-repaired, DSBs can result in cell death, mutations and cancer. In addition, defects in DSB repair (DSBR) underpin many other human diseases, including disorders associated with radiosensitivity, immune dysfunction, neurodegeneration and premature aging. Therefore, it is important to define all of the genes involved in DSBR. In human cells, many of the genes involved in DSBR have been identified during the last decade, frequently based on sequence homology to their respective orthologs in lower eukaryotes, such as yeast. Recently, however, it has become evident that DSBR pathways are much more complex in humans than in lower eukaryotes, and that lower eukaryotes do not encode all of the proteins involved in human DSBR. A new method has been developed to predict unknown gene function on the basis of inter-experimental behavior by using a global meta-analysis (GMA) of over 3,600 human 2-color microarray datasets in combination with literature data-mining software. Based on this approach, a novel human and apparently vertebrate-specific gene, tentatively named DNARR1 (DNA Repair Related 1), has been predicted and determined to play a role in DSBR. Such a role could not have been predicted on the basis of sequence homology. Strong experimental evidence suggests that DNARR1, like the breast cancer susceptibility genes BRCA1 and BRCA2, is involved in DSBR by homologous recombination, an essential pathway with known tumor-suppressor function. Since DNARR1 is located chromosome 9q21.13, a region of allelic imbalance in several types of cancers, this gene, like many other HRR genes, may also represent a new cancer susceptibility locus. In Aim 1 of this proposal the role of DNARR1 in homologous recombinational DNA repair (HRR) will be investigated. In particular, it will be determined if DNARR1 functions before or after IR-induced RAD51 focus formation, if DNARR1 binds to DNA and which HRR proteins DNARR1 interacts with. Using GMA to identify potential DSBR genes, DNARR1 is the only gene tested so far. However, the GMA has predicted 15 other novel DSBR genes, and these have been narrowed down to the six that we believe have the highest potential to be involved in DSBR. In Aim 2 these six genes will be investigated for their role in DSBR using in-vitro methods of genetic disruption. Specifically, it will be tested if functional loss of any of these six genes sensitizes human cells to IR (i.e. non-homologous end-joining) or drugs that interfere with DNA replication (i.e. HRR). For any new DSBR gene identified among these six, protein interaction partners will be determined and the ability to bind to DNA will be tested to further define its role in DSBR. This research, based on a unique approach to predict gene function, will expand our knowledge of human DSBR- associated genes and identify new disease susceptibility candidates. PUBLIC HEALTH RELEVANCE: DNA damage is a fairly frequent event in normal human cells, that have mechanisms in place to repair this damage when needed, even when the damage is severe enough to cleave the DNA in half via a double- stranded break (DSB), and defects in the repair of DSBs are responsible for many human diseases, the most frequent being a predisposition to cancer, but also radiosensitivity, immune dysfunction, neurodegeneration and premature aging. Here, a novel computational approach has predicted the involvement of seven new human genes in DSB repair by integrating a meta-analysis of co-expression patterns with large-scale literature data-mining. We have validated the role for the first of these genes in BRCA1/2-related DSB repair by homologous recombination (HR), and, in this grant proposal, will further define the exact role of this gene in HR, as well as test and further study the role of the other six genes in DSB repair.